Process for producing unsaturated steroids

ABSTRACT

An optically active compound having a 13 Beta -carbonsubstituted-17-hydroxy-8,14-secogona-1,3,5(10),9(11),15-pentaen14-one nucleus is produced from a compound having a 13 Beta carbon-substituted-8,14-secogona-1,3,5(10),9(11),15-pentaene14,17-dione nucleus by the action on the latter of the redoxenzyme system obtained by culturing a Candida, Debaryomyces, Kloeckera, Pichia, Rhodotorula, Schizosaccharomyces or Hansenula micro-organism.

United States Patent Masao Isono Nisliinomiya;

Takeshi Takahashi, Suita; Yoshio Yamasaki, Takarazuka; Takuichi Miki, Amagasaki, all of Japan [72] Inventors [2]] Appl. No, 746,882

[22] Filed July 23,1968

[45] Patented Oct. 26, 1971 [73] Assignee Takeda Chemical Industries, Ltd. Osaka, Japan [32] Priority July 24, 1967 [3 3 1 Japan [54] PROCESS FOR PRODUCING UNSATURATED STEROIDS 4 Claims, 11 Drawing Figs. [52] U.S.C| 195/51 R, 260/586 Primary Examiner-Alvin E. Tanenholtz Attorneywenderoth, Lind & Ponack ABSTRACT: An optically active compound having a 13,8-carbon-substituted-l 7-hydroxy-8, l 4-secogona- 1,3,5( l0),9( l l ),l5-pentaen-l4-one nucleus is produced from a compound having a l3B-carbon-substituted-8,l4-secogona- 1,3,5(10),9(1 l),l5-pentaene-14,17-dione nucleus by the action on the latter of the redox-enzyme system obtained by culturing a Candida, Debaryomyces, Kloeckera, Pichia, Rhodotorula, Schizosaccharomyces or Hansenula micro-organism.

PATENTEDUU 2 6 l9?! SHEET 10F 3 I L 350 400 WAVE LENGTH (M211 F 16 ID 350 400 300 WAVE LENGTHQW) 350 400 300 WAVE LENGTHWZA) FIGIB O NOISHI-JdSICI AHVLOH 35c 300 WAVE LENGTH m1) NOISHHdSICI AER/1.09:1

INVENTORS MASAO ISONO TAKESHI TAKAHASHI YOSH 1O YAMASAK] TAKUICHI MIKI ATTORNEYS PAIENTEllum 25 Ian SHEET 2 OF 3 MM 350 400 WAVELENGTH A) FIG 1F ZOGKMEQQ m kOm 350 WAVE LENGTH (mu) FIGJE O ZOEKMEQQ m kOm INVENTORS MASAO ISONO M HA 2 A K AMM T IY H mm EHU 7 MYT Y B L m5 m ATTORNEYS PATENTEDum 26 um SHEET 30F 3 (24 HOURS) 350 WAVELENGTH (mm) 0 ZQmmmEQQ m HOm (12 HOURS) FIGZB WAVELENGTH w) FIG 2 A (48 HOURS) INVENTORS MASAO ISONO 40o TA KESHI TAKAHASHI YOSHIO YAMASAKI TAKUICHI MIKI 35o WAVELENGTH (ma) (36 HOURS) WAVELENGTH (ma z) FIG 2C FIGZD 7 BY MWZZ, M

ATTORNEYS PROCESS FOR PRODUCING UNSATURATED STEROIDS The present invention relates to a process for producing optically active unsaturated steroids.

More particularly, the present invention relates to a process 5 for producing an optically active compound having a l3fl-carbon-substituted-l 7-hydroxy-8 ,14-secogona-polyenl 4-one nucleus [hereinafter referred to as compound (")1 from a compound having a l3fi-carbon-substituted-8,14-secogona- 1,3,5( 10), 9(l l),lS-pentaenel 4,1 7 dione nucleus [hereinafter referred to as compound (l)] through the utilization of a micro-organism.

Recently, methods for the total syntheses of l9-nor steroids have been developed. (See, for example, Miki, et al.: Proceedings of the Chemical Society 1963, p. 139; Crispin et al.: ditto, 1963, p. 22; Windholz et al.: Journal of Organic Chemistry, Vol. 28, p. 1092; and Smith et al.: Experientia, Vol. 19, p. 394.).

However, those methods are, commercially speaking, disadvantageous in that when a compound having a l3B-carbon substituted-EM4-secogona'polyene-l4,l7-dione nucleus is subjected to a ring-closing reaction to give the corresponding 1 3B-carbon-substituted-gonapolyenl 7-one nucleus, there arises a racemate with respect to the iii-substituent, resulting in a yield at best as low as only a half of the stoichiometric yield and, also, in that the steroid of natural type (where the Iii-substituent is B-oriented) is attained only on optical resolution.

Furthermore, two chemical methods for producing a comound having a lBfi-carbomsubstituted-l7a-hydroxy-8,l4- secogona-l,3,5(l),9(l l)-tetraen-l4-one nucleus have been established by Miki (one of the present inventors) and his coworkers, and by CR. Kuo et al. (Chemistry and Industry 1340, (1966)), respectively. It is understood, however, that the compounds attainable by those methods are invariably 50:50 mixtures of a compound having a lElfi-carbon-substituted-l7a-hydroxy-8, l 4-secogonal,3,5(l0),9( l l) -tetraen-l4-one nucleus and its enantiomer, and in order to convert them into natural estrone and the like, it is necessary to subject them to an optical resolution at some stage.

The main object of the present invention is to provide a novel and industrially feasible process for producing compound (ll) directly from compound (I) in a good yield.

The object ofthe present invention is realized by incubating a micro-organism belonging to the genus Candida, Debaryomyces, Kloeckera, Pichia, Rhodotorula, Schizosaccharomyces or Hansenula, contacting the redox-enzyme system of the thus-obtained culture with a compound having a l3,B-carbonsubstituted-8,l4-secogona-1,3,5( l0),9(] l),l5-pentaenel4,l 7-dione nucleus, and recovering the objective compound having a l3fl-carbon-substituted-l7-hydroxy-8,14-secogonapolyenl 4-one nucleus from the reaction mixture.

As the substituents at the l3-carbon, there may be exemplified lower alkyl, such as methyl, ethyl, propyl, butyl; aryl, such as phenyl; aralkyl such as benzyl, phenethyl, phenyl propyl, etc.

The compound (I) employed as the starting material, which can be produced, for example, by the method described in the Netherlands Pat. application No. 6,612,205 (published on Mar. 1, 1967), has a substituent at the l3-carbon and one to three other substituents-oxygen groups, halogen atoms and lower alkyl groups-at any of the l-,2-,3-,4-,6-,7-,1l-,l2-,l5- and lo-positions.

The oxygen groups include, for example, the hydroxy group, a lower acyloxy group containing one to four carbon atoms, an alkoxy group having one to four carbon atoms, and the benzyloxy group while the halogens include fluorine, chlorine and bromine, for instance. The lower alkyl groups are straight chain of one to four carbon atoms.

Some examples of the starting compounds of this invention are as follows:

8,14-secoestra-l ,3,5( l0),9( l l ),l5-pentaene-l4,l7-dione;

3-methoxy-8, l 4-secoestral ,3,5( l0),9(l l), l S-pentaene- 14,] 7-dione;

3-ethoxy-8, l4-secoestra-l,3,5( l0),9(l l),lS-pentaene- 3-methoxy-l3-ethyl-8,l4-secogona-l ,3,5( l0),9(l l ),15-

pentaene-l4,17-dione;

3-ethoxy-l 3-ethyl-8,l4-secogona-l ,3,5( l0),9( l l),l5-pentaenel 4,17-dione;

Spentaenel 4, l 7-dione;

pentaenel 4, l 7-dione;

3-benzyloxy-8,l4-secoestra-l ,3,5( l0),9(] l), l S-pentaene- 14,17-dione; 3-methoxy-l3-benzyl-8,l4-secogona-l,3,5( l0),9( l l ),l5

pentaenel 4, l 7-dione;

pentaene-l4,17-dione;

S-methoxy-l 3-phenyl-8, l 4-secogona-l ,3,5( l0),9(] 1), l5-

pentaenel 4, l 7-dione; 3-ethoxy l 3-phenyl-8,l4-secogona-l,3,5( l0),9( l l ),15-

pentaene-14,l7dione;

2,3-dimethoxy-l 3-ethyl-8,l4-secogona-l,3,5( l0),9( l l ),l

5-pentaene-l4,l7 dione; V 3-methoxy-6-methyl-8,l4-secoestras l ,3,5( l0),9( l l ),l 5

pentaene-l4,17-dione; and 3-methoxy-6,6-dimethyl-8,l4-secoestra-l ,3,5( l0),9( l l ),l

S-pentaenel 4, l 7-dione.

The objective compounds (II) can be classified as follows:

1. Compounds having a ISB-carbon-substituted-l 7B- hydroxy-8,l4-secogona-l,3,5( l0),9(l l), l S-pentaen-M-one nucleus, which are produced by such micro-organisms as Debaryomyces vini, Debaryomyces vanriji, Pichia wicherhamii, Pichia pijperi, Hansenula holstii and Hnnsenula capsulara.

2. Compounds having a l3B-carbon-substituted-l 7ahydroxy-8,l4-secogona-l,3,5( l0),9(l l),l 5-pentaenl 4-one nucleus, which are produced by such micro-organism as Kloeckera magna.

3. Compounds having a ISB-carbon-substituted-l7B- hydroxy-8, l 4-secogona-l ,3,5( l0),9(] l) -tetraen-l4-one nucleus, which are produced by such micro-organisms as Candida utilis, Debaryomyces nicatianae, Pichia etchellsii, Rhodomrula rubra, Schizosaccharomyces pombe, Hansenula sarurnus, Hansenula holstii.

The nutritive media for the growth of such micro-organisms contain the sources of carbon and nitrogen which can be utilized by the said micro-organisms and they may contain inorganic salts, various vitamins, and amino acids.

More specifically, the carbon sources include, for example, glucose, sucrose, dextrin, glycerol and others and the nitrogen sources include, for example, such organic nitrogenous materials as peptone, meat extract, casein, corn steep liquor, dry yeast and yeast extract and such inorganic nitrogen compounds as ammonium nitrate, ammonium phosphate, ammonium sulfate, and the like.

The above-mentioned inorganic salts include sodium chloride, potassium sulfate, magnesium sulfate and the like. Those nutrients promoting the growth of micro-organisms are employed in suitable proportions to make up a culture medium.

The cultivation may be carried out by any of the shake culture, the stationary culture and the submerged culture methods with agitation and aeration.

Addition of starting compound (I) can be made either at the start of the cultivation or at a suitable stage in the course of the cultivation, continuously or at some intervals or at one stroke, the final concentration of the compound (l) in the medium being advantageously between about 0.05 percent and about 0.6 percent (weight by volume basis).

The compound (I) can be employed in the form of a powder or as a solution or suspension in a suitable solvent, which may be acetone, methanol, ethanol, ethylene glycol, propylene glycol, dimethylformamide or dioxane, for instance, with or without addition of a surface active agent, dispersing agent and the like.

When the micro-organism is employed after separation from the culture liquid, the; microbial cells can be suspended in a buffer solution of suitable pH and ionic strength, or in water, and the starting compound (I) contacted therewith so as to convert the latter to compound (ll).

Usually the reaction proceeds at a pH from about 2 to about 11, and at a temperature somewhere between about 10 and about 50 C., advantageously between about 25 and about 40 C. for about 2 days.

However, the optimum conditions vary with such factors as the starting compounds and the micro-organisms and the optimum conditions are selected in each particular situation.

The final compound (ll) thus produced and accumulated in the reaction medium can be isolated by various per se known means.

By way of illustration, such separation means are available as adsorption by which the end product is adsorbed on a suitable adsorbent, e.g. silica gel, followed by elution with a suitable solvent such as benzeneacetone mixture or chloroformacetone mixture the means utilizing the difference in distribution coefficient between two liquid phases, for example, counter current distribution techniques, and conventional chromatographic techniques.

The optically active compounds with a l3B-carbon-substituted-l7-hydroxy-8,l4-secogona-1,3,5( l),9( l l ),l5-pentaen-l4-one nucleus are converted to the corresponding l,3,5( l0),bh9( l l) -tetraenes by the use of the enzyme system of a yeast, for example, Candida solani, after the manner of the process of the present invention.

Among the optically active compounds having a 13B-carbon-substitutedl 7-hydroxy-8, l 4-secogona-l ,3,5( l0),9(l l) tetraen-l4-one nucleus prepared above, the compounds having a l 3fl-carbon-substituedl 7a-hydroxy-8, l4-secogonatetraen-l4-one nucleus are converted to estrone or other useful l9-nor steroids for example, by the method described in French Pat. No. 1,526,031.

On the other hand, the compounds having a 13B-carbonsubstitutedl 7B-hydroxy-8, l 4-secogona-tetraenl 4-one nucleus are, for example, firstly subjected to cyclization in benzene with p-toluene sulfonic acid to give the optically active compounds having a 13B-carbon-substituted-l7B-hydroxy-gona-l,3,5(lO),8,l4-pentaene nucleus, which are then led to estrone through a series of the reactions disclosed in British Pat. No. 1,064,01 l.

Presently preferred embodiments of the invention are shown in the following examples, but these are not to be construed as limitative of the present invention.

, Throughout the specification, the abbreviation C. means degrees centigrade; percentages are calculated on the weight by volume basis unless otherwise noted.

The relationship between part(s) by weight and part(s) by volume is the same as that between gram(s) and milliliter(s).

Each of the specific micro-organisms employed in the following examples is available at Institute for Fermentation, Osaka, Japan, with reference to the respective accession numbers which are referred to as IFO-Numbers in the examples and those micro-organisms identified with N.R.R.L. number are available at Northern Regional Research Laboratory, Peoria, lll., while those with ATCC numbers are available at American Type Culture Collection, Rockville, Maryland.

EXAMPLE 1 Forty parts by volume of a liquid medium (pH 5.8) containmg:

0.3 percent of yeast extract, 0.5 percent of polypeptone, 0.2 percent of corn steep liquor, 5 percent of glucose and 5 per cent of sucrose are inoculated respectively with the 7-day slant culture of a micro-organism shown in table 1.

The inoculated medium is incubated at 28 C. for 2 days, at the end of which period there is added thereto a solution of 0.02 part by weight of 3-mcthoxy-8,l4-secoestra-1,3,5( l0),9( l l ),l5-pentaene-l4,l7-dione [hereinafter referred to as substrate (l)] in 0.8 part by volume of ethanol. The incubation is continued for further 2 days.

Then the resulting broth is mixed twice together with an equal amount of ethyl acetate, and the reaction products transferred to the ethyl acetate phase are washed with water, dehydrated and concentrated to dryness..

Column chromatography is carried out using silica gel, and the reaction products are isolated from the fraction eluted out with a 9:1 mixture of benzene and acetone. The products are assayed based on their rotatory dispersion curves in ethanol. FIGS. 1 (A) to (G) show the spectra of these compounds and of other steroids closely related thereto: (A) 3-methoxy-8,l4- secoestral ,3,5( l0),9(1 l) -tetraenl 7B-0ll 4-one (designated 183 in table 1 (B) 3-methoxy-8, l4-secoestra-l ,3,5( l0),9(] l ),l5-pentaen-l7B-ol-l4-one (designated A' -BB in table 1 (C) 3-methoxy-l3a-methyl-8,l4-secogona-l,3,5( l0),9( l l -tetraenl 7B-oll 4-one;

(D) 3-methoxy-1 3a methyl-8,14-secogona-l ,3,5(l0),9(l l), 15-pentaen-17B-0l-l4-one;

(E) a mixture of 3-methoxy-8,l4-secoestra-l,3,5(l0),9(l1 )-tetraen-l7B-ol-l4-one and 3-methoxy-8,l4-secoestra-l,3,5 (l0),9(AQ1l),l5-pentaen-l7B-ol-l4-one;

(F) a mixture of 3-methoxy-l3a-methyl-8,l4-secogonal,3,5( l0),9(1 l )-tetraem-l 7B-o1-l4-one and 3-methoxy-l 3amethyl-8,14-secogona-l ,3,5,( l0),9,l5-pentaen-l 7,8-ol-l4- one, and

(G) 3-methoxy-8,l4-secoestra-l ,3,5( l0),9(] l),l5-pentaen-l7a-ol-l4-one (designated A""-Ba in table 1), respectivey Whether the steric orientation between the l3-methyl and the l7-hydroxy is cis or trans can be judged by the test wherein the 8,14-seco-steroid compound is allowed to stand in methanol along with a small amount of p-toluene-sulfonic acid at room temperature, whereby the l3,l7-trans-compound gives a 9,14-oxide while the 13 l 7-cis-compound does not.

This test applies to the identification of the samples which can not be distinguished from each other on the basis of FIGS. 1, e.g. 3-methoxy-8,14-secoestra-l,3,5( l0),9( l l ),l5-pentaenl 7B-0ll 4-one and 3-methoxy l 3a-methyl-8, l 4- secogona-l,3,5( l0),9(! l ),l5-pentaen-17fi-ol-l4-one.

TABLE 1 UFO No.) Conversion Micro-organism NRRL Nov Product ratio or ('17 on weight ATCC No. basis) Candida un'lir (I086) pp 72 Debaryomyce: nicon'anae (0855) B3 75 ATCC 20l24 Debaryamyces vim (l2l4) A BB 68 Debaryomyce: vanriji U285) A' BB 70 ATCC 20125 Kloeckera magna (0868) A'Hsa 7s ATCC 20! 31 Pichin wickerllamii (I278) A fifl 65 Plchl'a etchellsii 1283) BB 70 ATCC 20l26 Plelu'a pijperl (I290) A' -flB ATCC 20l27 Rhodolaruln rubra (089 l) BB 75 ATCC (0362) ATCC (0980) NRRL Y-ZISS Hansenuln holsrii BBA' 'BB Hansznula salurnus (0993) ATCC EXAMPLE 2 One thousand parts by volume of a liquid medium (pH 5.8) containing 0.3 percent of yeast extract, 0.5 percent of proteosepeptone (Difco), 0.2 percent of corn steep liquor, 5 percent of glucose and 5 percent of sucrose are inoculated with a culture of Debaryomyces nicotianae (lFO-0855; ATCC 20124) and the micro-organism is incubated at 28 C. for 2 days. Then, a solution of one part of substrate (I) in 40 parts by volume of ethanol is added to the culture, and the incubation is continued for further 2 clays. The reaction product is extracted twice with 1,000 parts each by volume of ethyl acetate and, after the solvent is distilled off, is purified by column chromatography on silica gel with benzene-acetone mixture as eluant. The solvent is distilled off from the fraction containing the product and the residue is crystallized from ethanol to give 0.42 part by weight of 3-methoxy-8, 1 4-secoes tra-1,3,5(10),9(l l) -tetraen-l 7B-ol-l4-one as colorless plates melting at 112 to 113 C. and showing the negative Cotton effect curve and [a],, 39 (C=0.5 percent in dioxane).

EXAMPLE 3 1n the same manner as in example 2, a solution of one part by weight of substrate (1) in 40 parts by volume of ethanol is added to 1,000 parts by volume of a 2-day culture of Pichia elchellsii UFO-1283; ATCC 20126) which is then incubated for further 2 days. The reaction product is extracted withethyl acetate, purified by column chromatography and allowed to crystallize from ethanol to give 0.52 part by weight of 3- methoxy-8,l4-secoestra-l,3,5( l),9(l l) -tetraen-l7B-ol-l4 one as colorless plates melting at 11 1 to 113 C. and showing [a] ""39 (C =1 .0 percent dioxane).

EXAMPLE 4 In the same manner as in example 2, eight parts by volume of 2.5 percent solution of substrate (I) in ethanol is added to 200 parts by volume of a 2-day culture of Hansenula holstii 1FO-0980 which is then incubated at 28 C. The sampling is made at 12, 24, 36 and 48hours after addition of the substrate (1), and the samples are respectively subjected to extraction with ethyl acetate. The extracts are purified by column chromatography on silica'gel and are dissolved in ethanol.

The rotatory dispersion of the ethanolic solution is measured to give the results as shown in FIG. 2 (A,B,C, and D).

Those rotatory dispersion curves vary with the cultivation (reaction) time. Thus, the curves for the products corresponding to 12, 24 and 36 as well as 48 hours of cultivation are in agreement with those for 3 -methoxy-8,l4-secoestra-l,3,5( l0),9(l 1),] 5-pentaen-l7B-oll4-one, a mixture of 3-methoxy-8,14-secoestra-l,3,5( 9(1 I), -pentaen-l7/301-l4- one and 3-methoxy-8,l4-secoestra-l,3,5(10),9(1 l)-tetraenl7/3-0l-l4-one, and 3-methoxy-8,l4-secoestra1,3,5(l0),9( l 1) -tetraen-17B-ol-14-one, respectively,

While the product corresponding to the 12-hour reaction does not crystallize, its infrared absorption spectrum as measured in chloroform solution gives the absorption of hydroxy (3,620 cm"), and afi-unsaturated ketone (1,680 cm).

The rotatory dispersion of this product, which is reproduced in FIG. 2A, gives the negative Cotton effect curve having a bottom at 360 mu. Since no 3-methoxy-9,l4-oxido-8,14- secoestra-l,3,5(l0),9(1l) -pentaen-l7-one nor its enantiomer is formed when this product is treated with a small amount of p-toluenesulfonic acid in benzene, it is clear that the product is 3-methoxy-8,l4-secoestra'l,3,5(10),9(1l),l5 pentaen-l-7B-ol-l4-one.

The product attained after 36 and 418 hours of cultivation, respectively, is crystallized from ethanol in the form of colorless plates melting at 111 to 113 C. (Yield 78 percent on weight basis). The [01],, and infrared absorption spectra of the products are in perfect agreement with those of 3-methoxy-8,14-secoestra-1,3,5(10),9(11)-tetraen-17/3-o1-l4-one.

From the above, it is concluded that Hansenula holstii IFO-0980 has the property to first reduce the 17-carbonyl group of substrate (1) to yield 3-methoxy-8,l4-secoestra-l ,3,5 (l0),9(l 1),l5-pentaen-17B-ol-14-one and then reduce the A of the latter to yield 3-methoxy-8,14-secoestra-l,3,5 (10),9(1l)-tetraen-l7A-ol-14-one.

EXAMPLE 5 In the same manner as in example 2, a solution of 0.5 part by weight of substrate (1) in 40 parts by volume of ethanol is added to 1000 parts by volume of a 2day culture of Debaryomyces vanriji (IFO-l285; ATCC 20125) which is then cultivated as such for 2 days. The reaction product is extracted with ethyl acetate and the extract is purified by column chromatography after the manner described in example 2 to give 0.35 part of 3-methoxy-8,l4-secogona-l,3,5(10),9(l1),l5- pentaen-l 7B-ol-l4-one as an oil.

The rotatory dispersion of the oily product dissolved in ethanol solution is identical with the one reproduced in FIG. 1B. No. 9,14-oxide occurs when the product is treated with ptoluenesulfonic acid.

EXAMPLE 6 In the same manner as in example 2, 40 parts by volume ofa 2.5 percent solution of substrate (1) in. ethanol is added to 1,000 parts by volume of a 2-day culture of Kloeckera magna UFO-0868; ATCC 20131) and the reaction is allowed to proceed at 28 C. for 2 days, at the end of which period the product is extracted with ethyl acetate.

The extract is purified by column chromatography with silica gel after the manner described in example 2 to give 3- methoxy-8,l4-secoestra-l,3,5( l0),9,5-pentaen-l 7a-oll 4- one as an oil (yield 75 percent on weight basis),

The nuclear magnetic resonance spectrum of the productin CDC]; (Dzdeuterium) displays characteristic peaks of the A- l7-ol structure: pl l7-H (r 5.52, doublet), 16-l-l(r 2.62 quartet) and 15-1-1 ('r 3.89 quartet).

The rotatory dispersion of the product measured in an ethanol solution gives the positive Cotton effect curves as in FIG. 1 (G). The first peak occurs at 360 m=. This product gives a 9,14-oxide upon treatment with p-toluene-sulfonic acid in benzene.

What we claim is:

1. A process for producing an optically active compound having a l3B-carbon-substitutedl 7-hydroxy-8,l4-secogonapolyen-l4-one nucleus, which comprises subjecting a compound having a 13/3-carbon-substituted-8,l4-secogonal,3,5(l0),9(1l),l5-pentaene-l4,l7-dione nucleus to the action of an enzyme system of Candida utilis, Debaryomyces vini, Debaryomyces vanriji, Debaryomyces nicotianae, Kloeckera magna, Pichia wickerhamii, Pichia pijperi, Pichia etchellsii, Rhaa'otorula rubra, Hansenula capsulala, Hansenula holslii or Hansenula saturnus', and recovering the objective product from the reaction mixture.

2. A process according to claim 1, wherein the enzyme system is that of Debaryamyces vini IFO 1214, Debaryomycer (l0),9(AQl l ),l5-pentaen-l4-one nucleus.

4. A process according to claim 1, wherein the enzyme is that of Candida utilis [F0 1086, Debaryomyces nicon'anae [F0 0855, Pichia etchellsii [F0 1283, Rhodotorula rubra [F O 089 l Hansenula saturnus [F0 0993 orHanrenuIa holstii [F0 0980 (NRRL Y-2l55), whereby the obtained optically active compound is a compound having a lBB-carbon-substituted-i7phydroxy-8,l4-secogona-l ,3,5( l0),9(l 1 )-tetraen-14-one nucleus. 

2. A process according to claim 1, wherein the enzyme system is that of Debaryomyces vini IFO 1214, Debaryomyces vanriji IFO 1285, Pichia wickerhamii IFO 1278, Pichia pijperi, IFO 1290, Hansenula capsulata IFO 0984 (NRRL Y-1842) or Hansenula holstii IFO 0980 (NRRL Y-2155), whereby the obtained optically active compound is a compound having a 13 Beta -carbon-substituted-17 Beta -hydroxy-8,14-secogona-1,3,5(10),9(11),15-pentaen-14-one nucleus.
 3. A process according to claim 1, wherein the enzyme system is that of Kloeckera magna IFO 0868, whereby the obtained optically active compound is a compound having a 13 Beta -carbon-substituted-17 Alpha -hydroxy-8,14-secogona-1,3,5(10),9(11),15-pentaen-14-one nucleus.
 4. A process according to claim 1, wherein the enzyme is that of Candida utilis IFO 1086, Debaryomyces nicotianae IFO 0855, Pichia etchellsii IFO 1283, Rhodotorula rubra IFO 0891, Hansenula saturnus IFO 0993 or Hansenula holstii IFO 0980 (NRRL Y-2155), whereby the obtained optically active compound is a compound having a 13 Beta -carbon-substituted-17 Beta -hydroxy-8,14-secogona-1,3,5(10),9(11)-tetraen-14-one nucleus. 